PROJECT SUMMARY Hub proteins control cellular process by engaging in multiple protein-protein interactions (PPIs). These interactions are exceptionally challenging to target, in part due to the dynamic or disordered regions of the proteins that form the low to moderate affinity PPIs or ?fuzzy? interactions. A particular class of hub proteins, transcriptional coactivators, form PPIs with activator proteins and are critical for gene transcription. Mutation or misregulation of coactivators and their interactions lead to numerous diseases. Despite significant efforts in the field, coactivators have largely eluded small molecule targeting. The lack of molecular recognition principles for targeting this class of proteins is a substantial roadblock to successful and efficient ligand design and screening strategies. In this proposal, Tethering, a site-specific and covalent ligand discovery strategy, will be utilized to develop chemical co-chaperones or ligands that lead to enhancement or inhibition of PPIs. Chemical co- chaperones will be discovered for two model coactivators: dynamic Med25 and intrinsically-disordered SRC-3. My preliminary data suggest that coactivators can specifically recognize small molecules that contain PPI- privileged substructures. These substructures have distinct geometries, pre-organized conformations, and localized electronic properties similar to natural products, a rich source of modulators for PPIs. Building on my preliminary data, we hypothesize that a rationally designed PPI-focused Tethering library and an activity-based screening strategy can lead to molecular recognition frameworks that describe the affinity, activity, and selectivity of coactivator:chemical co-chaperone interactions for the first time. In Aim 1, a PPI-focused Tethering library will be rationally designed and synthesized utilizing commercially available building blocks of known PPI-privileged substructures or natural product-based fragments. A proof-of-concept screen will be conducted to ensure known molecular patterns are identified for Med25. In Aim 2, intact mass spectrometry (MS) will be utilized to conduct an activity-based Tethering screen for Med25:activator interactions. The lead enhancers and inhibitors will then be utilized in Extension Tethering, a fragment growing strategy. In Aim 3, Tethering, Extension Tethering, and intact MS strategies will be utilized to identify chemical co-chaperones for SRC-3:nuclear receptor interactions. SRC-3 is a particularly interesting hub as it lacks defined 3D structure and contains multiple reactive cysteines. In each of these aims, molecular recognition frameworks will be constructed by statistical analysis of 2D and 3D descriptors and by developing 4D-Quantitative Structure-Activity Relationships, where the features and topology of the binding site is mapped. Further, the chemical co-chaperones identified will be utilized to study the unique biophysical and structural properties of dynamic Med25 and for the first time, intrinsically disordered SRC-3. These frameworks will be utilized to determine the critical features of PPI recognition for building and enriching PPI-targeted small molecule libraries. We expect the frameworks and guiding principles discovered herein will be generalizable to other ?undruggable? hub proteins, which have extraordinary promise as therapeutic targets.